Detection of nucleic acid variants is important with respect to a variety of situations, and critically important with respect to detection and prognosis of diseases. There are currently a wide range of assay formats for detecting nucleic acid variants. Such assays include pyrophosphorolysis-activated polymerization (PAP), assays using LNA blockers, and cast-PCR assays.
Pyrophosphorolysis-activated polymerization (PAP) can be used to measure mutation load or to detect minimal residual disease. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially by utilizing a pyrophosphorolysis-activatable oligonucleotide (P*). The activated P* can be extended by DNA polymerization. Specificity of the assay results from both pyrophosphorolysis and polymerization as significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, which is an extremely rare event. (See, e.g., Liu and Sommer, “Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification,” Biotechniques, 29(5):1072-6, 1078, 1080 (2000); incorporated by reference herein in its entirety.)
LNA blockers can also be employed with methods of detecting and/or quantifying nucleic acid variants in populations of nucleic acids where the wild-type nucleic acids are in greater abundance. Such methods utilize short high affinity oligonucleotides targeted to the wild type rather than the minority or mutant sequence and which function to block detection of wild type DNA. The LNA blocker probes can be used in combination with longer detection probes or PCR primers to amplify and/or identify the minority or mutant sequence. (See, e.g., U.S. Patent Appl. No. 20100009355; incorporated by reference herein in its entirety.)
Cast-PCR can also be used as an assay for analyzing sequence variation between different alleles. The methods use competitive allele-specific TaqMan PCR (“cast-PCR”) to distinguish nucleic acid variants. Cast-PCR employs performing two amplification reactions on a target nucleic acid sequence. The first reaction includes amplification in the presence of a first allele-specific primer and a first allelic specific blocker, which is complementary to the first allelic variant, followed by detection of the amplification product. The second reaction includes amplification in the presence of a second allele-specific primer and a second allelic specific blocker, which is complementary to the second allelic variant, followed by detection of the amplification product. (See, e.g., U.S. Patent Appl. No. 20100221717; incorporated by reference herein in its entirety.)
All of the above methods possess various challenges and limitations. A common problem behind these approaches for detecting rare variants is poor enzyme fidelity. Errors introduced during replication and associated amplification, can not be easily discriminated from true rare variants and mutations, and thus undermine the performance of these approaches. Additionally, in the case of allele specific priming, such as the amplification refractory mutation system (ARMS, Nucleic Acids Res. 17:2503-16 (1989)) mispriming during amplification can “over-write” variant sites and lead to poor results. In most cases specificity of the above methods is limited to 0.1-5% of allele prevalence. This is the case even with next generation sequencing approaches, since the polymerases employed introduce errors sufficient to limit the sensitivity of detection of rare alleles to about 3-5%. A key difficulty with all of these assays is that at their core are amplification methods that use DNA polymerases which due to their infidelity introduce errors that create an intrinsic background in the 1-3% range or more, depending upon the polymerase. A few methods can give much higher levels of specificity, such as digital PCR, but these methods are complex, expensive, and do not interface easily to confirmatory or diagnostic assays. Additionally, digital PCR also does not lend itself to high levels of multiplexing.
There is a need in the art for additional assay methods that can more effectively detect rare nucleic acid sequence variants. Furthermore methods are needed that reduce incorporation errors associated with polymerase associated amplification systems. Additional assays need to be developed and the methods of the present invention provide such additional assay methods.